Clostridium difficile (C. difficile or C. diff) is a Gram-positive, spore-forming bacterium that causes hospital-acquired as well as community-acquired enteric infections. Infections can be asymptomatic but generally lead to C. diff-associated diarrhea, pseudomembranous colitis, colitis and death (Halsey, J:, 2008, Am. J. Health Syst. Pharm. 65: 705-715; Lessa, F. C. et al., 2012, Clin. Infect. Dis. 55: S65-70). Approximately 20% of individuals who are hospitalized become colonized with C. difficile during hospitalization, and more than 30% of these patients develop diarrhea. Thus, Clostridium difficile infection (CDI) or Clostridium difficile-associated disease (CDAD) is now a major problem in hospitals worldwide (Kutty, P. K., et al., 2010, Emerg. Infect. Dis 16: 197-204; O'Brien, J. A. et al, 2007, Infect. Cont. & Hosp. Epidemiology 28: 1219-1227.
The rate of CDI is steadily increasing both in North America and Europe (Goorhuis, A et al., 2008, J. Clin Microb. 46: 1157-1158; Gravel, D. et al., 2009, Clin. Infect. Dis. 48: 568-576). It is estimated that 1.1 to 3 million patients are infected with C. diff and there are 30,000 C. diff-related deaths each year in the US alone3. Similar numbers of people are affected with CDI in Europe, costing the EU health care an estimated $4.4 billion each year (Lessa, F. C. et al., 2015, N. Engl. J. Med. 372: 825-834).
CDI has surpassed methicillin-resistant staphylococcus aureus (MRSA) as the most frequent infections acquired in the hospital and controlling CDI has proven to be a challenge (Miller, B A. et al., 2011, Infect. Control Hosp. Epidemiol 32: 387-390). In the U.S., patients with C. diff are hospitalized for an extra 3.6-7 days, costing more than $8.2 billion (Lessa, F. C. et al 2015) (6; 7; 8.
C. difficile infection is often but not always induced by antibiotic disruption of the colonic flora through the use of antibiotics such as clindamycin, cephalosporins, and fluoroquinolones. This perturbation in the colonic microenvironment, along with exposure to C. difficile spores, leads to mucosal C. difficile colonization. This colonization may result from the presence of a pre-existing antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Approximately one-third of all patients that become colonized develop CDAD, which can result in severe diarrhea, perforation of the colon, colectomy and death. CDAD results from the acquisition and proliferation of C. difficile in the gut, where C. difficile bacteria produce toxin A and toxin B, two important virulence factors of CDAD. Toxins A and B of C. difficile show considerable sequence and structural homology. Both have a C-terminal receptor-binding domain containing multiple repeating sequences, a central hydrophobic domain and an N-terminal glucosyltransferase domain. The receptor-binding domain mediates binding of the toxins to intestinal epithelial cells via host receptors that remain poorly defined in humans. Following internalization via an endosomal pathway, the central hydrophobic domain inserts into the membrane of the endosome. The acidic pH of the endosome triggers pore formation and translocation of the amino-terminal domains of the toxins into the cytosol. Glucosylation of the cytosolic target Rho GTPases leads to disruption of the cytoskeleton and cell death. Toxins A and B demonstrate different pathological profiles and have potential synergy in causing disease.
Current treatment for C. difficile infection (CDI) is the use of certain antibiotics, such as for example but by no means limited to Metronidazole, Vancomycin, and Fidaxomycin, either alone or in combination. However, efficacies of these antibiotics are limited by incomplete response rates with increasing re-infection and recurrence rates2. The antibiotic therapy does not provide complete protection to all patients; as a result, 25-40% of patients suffer from C. difficile recurrent infections (Figueroa, I. et al., 2012, Clin. Infect. Dis. 55: S104-S109).
The risk of recurrence is increased in patients who have already had one recurrence, rising from 20% after an initial episode to more than 60% in patients with a history of previous C. difficile infection. Furthermore, C. difficile strains are becoming resistant to antibiotic therapies. As a result, cure rates are decreasing and the rate of recurring infections is increasing along with increased severity and mortality even with antibiotic therapy (Kelly, C. P. and Lamont, J. T., 2008, N. Engl. J. Med. 359: 1932-1940; Goorhuis, A et al 2008; Gravel, D. et al 2009).
The prevalence of C. difficile infection has been increasing steadily, particularly in the elderly, who are often frail. Approximately one-third of patients with a C. difficile infection have recurrences of their infection, usually within two months of the initial illness. Repeat infections tend to be more severe than the original disease. Older adults and people with weakened immune systems are particularly susceptible to recurring infections (Kee, V. R., 2012, Am. J. Geriatric Pharm. 10: 14-24). If not treated promptly and appropriately, the complications of C. difficile infection include dehydration, kidney failure, bowel perforation, toxic megacolon, which can lead to rupture of the colon, and death. C. difficile has become the most common cause of health-care associated infections in US hospitals. Health-care cost related to C. difficile infections are estimated to be as much as $4.8 billion for acute care facilities alone. In addition, C. difficile infection has been increasing reported outside the acute care facilities, including in community and nursing home settings, where infection can be treated without hospitalization. The elderly people who are at the high risk of getting C. difficile infection have other debilitating diseases like cancer, HIV, undergoing surgery, prolonged treatments with antibiotics, other gastrointestinal diseases.
The incidence and severity of CDI have increased significantly due in part to the emergence of unusually virulent, antibiotic resistant strains. Chief amongst these are strains characterized as group BI by restriction endonuclease analysis, North American pulse-field type 1 (NAP1) by pulse-field gel electrophoresis and ribotype 027 by polymerase chain reaction. These hyper-virulent strains are often also toxin-hyperproducers. For example, isolates of ribotype 027 produced higher levels of toxin and exhibited slower growth compared to other isolates (Figueroa, I. et al 2012).
These strains cause CDI with a directly attributable mortality more than 3 fold that observed previously.
Furthermore, isolates demonstrating increased spore production appear to be linked to more severe C. difficile infections (Merrigan, M. et al., 2010, J. Bacteriol. 192: 4904-4911; Carlson, P. E. et al., 2013, Anaerobe 24: 109-116).
Consequently, there is a need for more effective treatments that target the life-threatening diseases caused by C. difficile, and, in particular, the potent toxins that are produced by C. difficile, for prophylactic and therapeutic benefit. There is an unmet medical need for successful and lasting treatments for C. difficile-associated disease that offer lower potential for developing resistance and higher potential for successful patient response and disease resolution, leading to disease eradication.
Thus, it is clear that the disease/infection caused by C. difficile puts the lives of people of all ages in jeopardy, both in nosocomial settings and in the community at large. In today's world, there is an ever present risk of C. difficile infection for those who face hospitalization or who are in long-term care. New therapeutics are therefore urgently needed since efficacy of the current antibiotics appears to be decreasing. Consequently, there is a clear unmet medical need for an effective novel non-antibiotic therapy that targets the life-threatening disease caused by the “super bug”, C. difficile. The present invention meets this need and provides compositions and methods for safe, effective treatment and prevention of CDI or CDAD.
Immune approaches attempted so far include vaccination (Salcedo, J. et al., 1997, Gut. 41: 366-370), the use of anti-Clostridium difficile colostral or whey protein from cows immunized with C. difficile and passive immunization with intravenous immunoglobulin (IVIg) (Surawicz & Alexander, 2011, Nature Rev. Gastro. Hep. 8: 339-339), Despite some encouraging studies in a limited number of patients, neither specific cow antibodies nor polyclonal IVIg has become a commonly used treatment.
Vaccination offers the potential advantage of generating a polyclonal response, but the disadvantages relate to the timing of vaccine administration relative to the perceived risk of infection and variability in the patient's immune response.
Furthermore, the manufacture of colostrum or serum polyclonal antibodies from cow, sheep, alpaca or other species for therapeutic use can have production quality difficulties in controlling the variability of batch-to-batch antitoxin activities (titer and specificity). In addition, total production capacity of methods such as these have limits and there can be immunogenicity concerns.
U.S. Pat. No. 8,986,697 teaches monoclonal antibodies which bind to toxin A and toxin B of C. difficile. 
U.S. Pat. No. 8,257,709 also teaches monoclonal antibodies which bind to toxin A and toxin B of C. difficile. 
U.S. Pat. No. 8,921,529 teaches ovine antibodies raised against C. difficile toxin A, Toxin B and/or binary toxin which are administered as sheep serum.
Polyclonal antibodies comprise a large number of antibodies with different specificities and epitope affinities. For production purposes these antibodies are generally purified from the serum of immunised animals were the antigen of interest stimulates the B-lymphocytes to produce a diverse range of immunoglobulin's specific to that antigen.
Monoclonal antibodies represent a single B lymphocyte generating antibodies to one specific epitope.
Polyclonal antibodies are poly-specific in that a polyclonal antibody preparation is composed of many different antibodies, each recognising a distinct epitopes of one antigen. While in some uses, such as simple detection of a specific antigen, the fact that a polyclonal antibody preparation will bind to more than one epitope on a specific antigen is not an issue, this is generally considered to be a negative in therapeutic uses as many of the polyclonal antibodies are likely to recognize non-neutralizing epitopes and therefore may be of limited value from a therapeutic standpoint. Cross-reactivity can also be a concern.
Monoclonal antibodies are produced from an immortal cell line and therefore it is possible to produce unlimited quantities of highly specific antibodies. Furthermore, all batches will be identical and specific to just one epitope, which is generally considered to be a particular advantage for therapeutic treatments.
However, neutralizing monoclonal antibodies with high affinities, in vitro potencies or very high levels of protection against infection in most animal models can be very difficult to obtain.
Furthermore, many monoclonal antibodies which show successful treatment of a given disease in lower mammals have failed when used on higher primates for reasons that cannot always be explained.
Furthermore, polyclonal antibodies have a significant advantage in that they are less sensitive to antigen changes than monoclonal antibodies. As such, polyclonal antibodies are more tolerant to mutations and/or effective against different strains and/or isolates, by virtue of recognizing multiple epitopes. Specifically, polyclonal antibodies recognize multiple inhibitory epitopes on a single target and accordingly can achieve beyond what is possible against a single monoclonal antibody.
For example, while monoclonal antibodies against the cell wall binding domains of the C. difficile toxin proteins have demonstrated neutralizing capabilities, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures (Corthier, G. et al., 1991, Infect. Immun. 59: 1192-1195; D. F. M. Thomas et al., 1982, The Lancet, Jan. 9, 1982, pp 78-79; Lyerly, D. M. et al., 1985,J. Clin. Micro. 21: 12-14).
Clearly, a therapeutic composition for treating C. difficile infections and C. difficile associated diseases which has broad applicability, particularly against hypervirulent strains, that can be easily and reproducibly produced and will be well tolerated by patients suffering from CDAD is needed.